Pancreatic ductal adenocarcinoma (PDAC) is the most lethal of all human malignancies, remains virtually untreatable, and is increasing in incidence with every year. Because the landscape of driver mutations in PDAC is extremely narrow, it is likely that transformative improvements in the treatment of PDAC will occur only through fundamental breakthroughs in our understanding of the biology of established PDAC tumors. However, the field lacks tools necessary to study gene function in PDAC tumors that have arisen through the expression of endogenous oncogenic Kras in the pancreatic epithelium, which mimics PDAC development as it occurs in patients. Thus, the overall goal of this research project is to establish dual recombinase PDAC models that will permit studies of gene function in specific cell types, after tumor formation. These models will provide unparalleled access to the biology of PDAC tumors, ultimately permit studies of gene function in stromal cell types, and enable the validation of candidate PDAC essential genes and therapeutic targets in models of PDAC that most closely resemble human disease. While Cre/lox -dependent models recapitulate the genetics, histopathology, and molecular pathology of human PDAC, and develop stroma-mediated drug resistance phenotypes akin to that seen in patients, they lack an additional means to perform independent gene targeting. To solve this problem, we will establish Flp/FRT models of PDAC and identify ubiquitous CreER alleles that can be used to model therapy and assess global consequences of the loss of candidate PDAC essential genes, after tumor formation. In our first aim, we will establish Flp/FRT models of PDAC in which pancreas-specific Flp recombinase is supplied by a heritable Pdx1-Flp transgene (Aim 1A) and, to minimize the cost and time resources necessary to perform dual recombinase experiments, via adenoviral pancreas- specific Flp expression vectors (Aim 1B). The expression of Flp recombinase in the pancreatic epithelium will lead to the activation of an endogenous FRT-STOP-FRT-KrasG12D allele and to the deletion of a p53FRT allele, which will culminate in PDAC and permit Cre and lox alleles to be used subsequently for studies of gene function. Together these reagents will provide robust Flp/FRT models of PDAC development and maximize the accessibility of dual recombinase PDAC models to members of the PDAC research community. In Aim 2, we will identify ubiquitously expressed CreER alleles that most efficiently target all cells in Flp/FRT PDAC tumors and throughout the diverse tissues of the mouse. This will provide a dual recombinase system that, in conjunction with floxed alleles, can be used to model therapy and assess the global consequences of the loss of candidate PDAC essential genes, providing not only genetic preclinical efficacy data, but the opportunity to identify potential side-effects and toxicities associated with agents designed to target the proteins encoded by candidate PDAC essential genes.